The present invention relates to novel compounds and pharmaceutical preparations comprising same, their use in the treatment of and in the diagnosis of certain diseases, in particular of diseases involving changes of cell membrane lipid asymmetry
(CMLA) is the phenomenon, by which normal eukaryotic cells have an asymmetrical organization of the phospholipids comprising their plasma membranes; the outer membrane leaflet is formed predominantly with the cholinephospholipids: (phosphatidylcholine [PC] and sphingomyelin), whereas the majority of the amino phospholipids (phosphatidylserine [PS] and phosphoethanolamine [PE]) are confined to the membrane""s inner leaflet (Zwaal R F A and Schronit A J, Blood 1997;89:1121-1132) The physiolocical importance of CMLA is exemplified by the fact that its maintenance requires a continuous, considerable investment of energy by the cell (Seigneuret M and Devaux P F, Proc. Natl. Acd. Sci., 1984;81:3751) At least three distinct systems are active in the regulation of CMLA:
1. Aminophospholipid translocase (APT): an ATP-dependent enzyme which transports PS and PE from the outer to the inner membrane leaflet, against the concentration gradient (Daleke D L and Huestis W H, Biochemistry 1985;24:5406).
2. ATP-dependent floppase: transports amino-phospholipids and cholinephospholipids from the inner to the outer leaflet. This enzyme is tenfold slower than APT (Andrick C et al., Biochim. Biophys. Acta 1991;1064:235).
3. Lipid scramblase: A potent, Ca2+-dependent and ATP-independent enzyme, that rapidly moves phospholipids back and forth between the two membrane leaflets (flip-f-lo), leading within minutes to loss of CMLA (Zwaal R F A and Schronit A J, Blood 1997;29:1121-1132)
In addition, other factors, such as membrane anchoring of cytoskeletal proteins have been suggested to assist in CMLA maintenance.
Whereas the maintenance of CMLA is fundamental to normal cell physiology, its loss, with subsequent surface exposure of PS plays a role in numerous states of both physiological and pathological characters. The surface exposure of anionic phospholipids plays an indispensable role in the formation of a catalytic surface for the assembly of several clotting factor complexes. Thus, the loss of CMLA in activated platelets as well as in other cell types (e.g. endothelial cells), is an important factor in normal blood coagulation. However, CMLA loss also assists in the initiation and/or propagation of abnormal, excessive blood clotting in numerous disorders. These disorders include, among others:
1. Arterial or venous thrombosis (Thiagarajan P and Benedict C R, Circulation 1997;96:2339-2347; Van Ryn McKenna J, et al., Throm. Hemost. 1993;69:227-230).
2. Sickle cell disease (Tait J F and Gibson D, J. Lab. Clin. Med. 1994;123:741).
3. Beta-thalassemia (Borenstein-Ben-Yashar Y, et al., Am. J. Hematol. 1994;47:295; Ruf A, et al., Br. J. Haematol. 1997;98:51-56).
4. Antiphospholipid antibody syndrome; among others in systemic lupus erythematosus. Lack of CMLA has been specifically linked to the recurrent abortions associated with said syndrome (Rand J H, et al., N. Engl. J. Med. 1997;337:154-160).
5. Shed membrane microparticles, e.g., during cardiopulmonary bypass, (Nieuwland R et al., Circulation 1997;96:3534-3541; Aupeix K, et al., J. Clin. invest. 1997; 99:1546-155).
Apoptosis is another major situation in which CMLA loss takes place. Apoptosis is an intrinsic program of cell self-destruction or xe2x80x9csuicidexe2x80x9d, which is inherent in every eukaryotic cell. In response to a triggering stimulus, cells undergo a highly characteristic cascade of events of cell shrinkage, blebbing of cell membranes, chromatin condensation and fragmentation, culminating in cell conversion to clusters or membrane-bound particles (apoptotic bodies), which are thereafter engulfed by macrophages (Boobis A R, et al., Trends Pharmacol. Sci. 10:275-280, 1989; Bursch W, et al., Trends Pharmacol. Sci. 13:245-251, 1992). Loss of CMLA is quite a universal phenomenon in apoptosis (Van den Eljnde S M, et al., Cell death Diff. 1997;4:311-316). Loss of CMLA occurs early in the apoptotic cascade, immediately following the point of cell commitment of the death process (Van-Engeland M, et al., Cytometry 1998;31:1-9; Martin S J, et al., J. Exp. Med. 1995;182:1545-1556). It has also been shown that the loss of CMLA is an important factor in the recognition and removal of apoptotic cells by macrophages (Balasubramanian K, et al., J. Biol. Chem. 1997;272:31113-31117). A strong correlation has recently been drawn between the loss of CMLA and the potent pro-coagulant activity of apoptotic cells (Bombeli T, et al., Blood 1997; 89:2429-2442; Flynn P D, et al., Blood 1997;89:4378-4384) The latter activity in apoptotic endothelial cells, such as those recently recognized in atherosclerotic plaques (Kockx M M, et al., Circulation 1998;97:2307-2315, Mallat Z, et al., Circulation 1997;96:424-428), probably plays an important role in the pathogenesis of thrombotic vascular disorders.
The diagnosis of the loss of CMLA may therefore serve as an important tool for the detection of cell death, specifically by apoptosis. A method for the detection of cell death may have many applications, both as a diagnostic tool and as a method to monitor the disease course in numerous disorders associated with impairment of tissue homeostasis. Among these applications are:
1. Monitoring of a response to anti-cancer therapy:
Currently there is a lag period between the time of administration of anticancer drugs and the time of evaluation or their efficacy. Thus, in case of failure of a therapeutic regimen, this lag time may be hazardous to the patient in two aspects:
a. loss of precious time without an effective therapy; and
b. unnecessary exposure of the patient to drug adverse effects.
Therefore, there is clearly a need for an early detection of tumor response to treatment. Since anti-tumor drugs exert their effects by induction of apoptosis (Eastman A, Cancer Cells, 1990;2:275-280), the detection of apoptosis, potentially by detection of CMLA loss may be useful for monitoring tumor response.
2. Diagnosis of disorders of inappropriate excessive apoptosis. These disorders include, among others, AIDS, neurodegenerative disorders, myelodysplastic syndromes and various ischemic or toxic insults (Thompson C B, Science 1995;267:1456-1461).
3. Monitoring of graft survival following organ transplantation. The increasing use of organ transplantation for the treatment of end-stage organ failure emphasizes the need for the development of methods for sensitive monitoring of graft survival. Apoptosis plays a major role in graft cell loss (Matsuno T, et al. Transplant Proc. 1996;28;1226-1227; Dong C et al., Lab. Invest. 1996;74:921-931).
4. Monitoring of response to cytoprotective treatments. The current intensive research of cytoprotective agents, towards development of drugs capable or inhibiting cell loss in various diseases (Thompson C B, Science 1995;267:1456-1461), dictates a need for measures to evaluate the effects of such compounds, i.e., monitoring of cell death, in all levels of research, from in vitro tissue culture studies, through in vivo animal models to human clinical studies.
5. Basic research of apoptosis in tissue cultures and animal models.
The loss of the normal CMLA has, as indicated above, wide implications for various pathophysiological states. A compound capable of selectively binding to membranes upon CMLA loss, thus serving as a marker for this phenomenon, may therefore have wide diagnostic applications. Moreover, by shielding the exposed anionic phospholipids, specifically PS, such compound may be a useful therapeutic agent, for example for the above-mentioned disorders, which are associated with excessive pro-coagulant activity caused by the membrane phospholipid re-organization.
In addition, a compound capable of detecting cells undergoing apotosis may have important applications for targeting drugs to apotosis-inflicted tissues. Apoptosis and its major control systems are shared by all tissues in the body. Therefore, the implementation of the emerging new generation or drugs, active by modulation of apotosis control is expected to depend, at least in part, on the ability to target these drugs to the appropriate tissues. An apoptosis-detecting compound may thus be useful for this task.
There have been developed certain measures for the effective detection of cell death in tissue cultures, such as the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling) method, for the detection of the characteristic chromatin cleavage of apoptotic cells. However, this method, as well as other methods such as the DNA laddering method, are strictly limited to the in vitro level.
The potential of a detector of CMLA loss both as a diagnostic tool and as a therapeutic measure has recently been exemplified by the use of annexin-V for these indications. Annexin V is a member of the annexin family of proteins, sharing potent, Ca2+-dependent binding to anionic phospholipid membranes (Swairjo M A, et al., Nature Struc. Biol. 1995;968-974). Annexin V is a 320 amino acid protein, with a molecular mass of 35,935 daltons (Huber R, et al., EMBO J. 1990;9:3867-3874) Though the physiological role of annexin-V has not been fully elucidated, it has been suggested to be involved in anticoagulation, anti-inflammation and cellular signaling (Romisch J, et al., Thromb. Res. 1990;60:355-366; Bastian B C, J. Invest Dermatol. 1993; 101:359-363; Kaneko N, et al., J. mol. Biol. 1997;274:16-20). The impressive affinity of annexin V to anionic phospholipid membranes (Kd of about 10xe2x88x929-10xe2x88x9211M, [Hofmann A, et al., Biochim. Biophys. Acta, 1997;254-264]) has been extensively utilized for both the diagnosis of CMLA loss and modulation of disorders associated with this phenomenon. Fluorescein isothiocyanate (FITC)-labeled annexin V has been widely used for the detection of apoptosis in various tissue culture models (Koopman G, et al., Blood 1994;84:1415-1420; Rimon G, et al., J Neurosci Res 1997; 48:563-570; Van-Engeland M, et al., Cytometry 1998;31:1-8). Preliminary successful studies were also performed with systemic intracardial injection of biotinylated annexin v to viable mouse embryos, for the detection of developmentally-associated apoptosis (Van den Eijnde S M, et al., Cell death Diff. 1997;311-316). Systemic administration of 99mTc-annexin V was also used to detect and image cell death in several models in vivo, e g. fulminant hepatitis in mice, acute rejection of transplanted cardiac allograft in rats and monitoring of response of lymphoma to cyclophosphamide treatment in mice (Blankenberg F G. et. al. Proc. Natl. Acad. Sci. USA, 95:6349-6354, 998). 125I-labeled annexin V was also used for in vivo detection of thrombosis in an animal model (Stratton J R, et al., Circulation 1995;92:3113-3121). Inhibition of arterial thrombosis was effectively achieved by intravenous administration of annexin V in a carotid artery injury model (Thiagarajan P and Benedict C R, Circulation 1997;906:2339-2347). Annexin V is also known as diagnostic agent (U.S. Pat. No. 5,627,036).
However, the use of annexin V as a drug or as a diagnostic probe is rendered problematic by several characteristics of this protein. Annexin V is a protein of considerable size, a factor which may substantially limit its volume of distribution in the body. Moreover, it is active as a potent membrane-binding protein only if allowed to form a highly organized multimer on the membrane surface (Concha NO, et al., FEBS Lett 1992;314:159-162; Voges D, et al., J. Mol. Biol. 1994;238:199-213, Andree H A M, et a., J. Biol. Chem 1992;26:17907-17912). Thus, systemic administration of annexin V as a drug is expected to be associated with rapid degradation and loss of the function of the administered protein. Indeed, a very rapid clearance (90% within 5 minutes) was observed in rabbits following intravenous injection of annexin V (Thiagarajan P and Benedict C R, Circulation 1997;96:2339-2347). In addition, the administration of annexin V may induce an untoward immunological response, importantly, anti-annexin V antibodies have been recently implicated in the pathogenesis of anti-phospholipid antibody syndrome and associated thrombotic events (Nakamura N, et al., Am. J. Hematol. 1995; 49:347-348; Kaburaki J and Ikeda Y, Rinsho Ketsueki 1995;36:320-324, Rand J H, et al., N. Engl. J. Med. 1997;337:154-160).
There exists therefore a need for novel methods for the detection of cell death, specifically at the in vivo level. A method for the detection or loss of CMLA may be useful for this purpose.
Moreover, it is also desirable to develop novel compounds, for the diagnosis of CMLA loss, the modulation of its pathophysiological consequences and for the treatment of certain diseases in which said CMLA loss plays a role.
The present invention thus consists in a compound (hereinafter: xe2x80x9cNST300 compoundxe2x80x9d) of general formula I comprising the following components:
X1.[(X3)a/(X4)b]
wherein:
X1 stands for a saturated or unsaturated fatty acid residue comprising 6-20 carbon atoms; or a cysteine residue bound through a thioether bond to a prenyl group
comprising 5-20 carbon atoms, said residue being linked to the adjacent component of the compound through an amide bond;
X3, comprises 1-6 amino acids, of which 1-6 are positively charged, the other amino acid residues being polar uncharged amino acids; and
X4 comprises 1-6 amino acids, of which 1-2 are aromatic amino acids, the other amino acids being selected among polar uncharged amino acids and hydrophobic aliphatic amino acids;
xe2x80x83wherein:
a stands for an integer of 1-8; and
b stands for an integer of 1-8;
the groups X3 and X4 being located at various places in the compound.
For the sake of clarity it should be indicated that the term xe2x80x9cprenylxe2x80x9d herein, stands also for the term xe2x80x9cisoprenylxe2x80x9d (see Stedman""s Medical Dictionary, Baltimore, USA, William and Wilkins, eds., 1990:565, 1253).
X1 serves as main anchoring domain A;:
X3 serves as anionic phospholipid binding determinant; and
X4 serves as accessory anchoring domain.
X0 is advantageously a residue of a saturated fatty acid of formula CH3(CH2)nCO2H, in which n stands for an integer of 8-18 preferably selected among myristic acid and palmitic acid; or X1 is advantageously a cysteine-residue bound through a thioether bond to a prenyl of 5-15 carbon atoms, preferably farnesyl cysteine.
The positively charged amino acids of X3 are advantageously selected among lysine, arginine, histidine or any amino acid which is comprised of a positively charged group, e.g. primary amine, secondary amine, guanidine, covalently bound to the xcex1-carbon atom or to the xcex1-amine on the peptide backbone bad a spacer comprised of an alkene of 1-4 carbon atoms; and combinations thereof; the other amino acids that are not positively charged are polar uncharged. The acids are preferably selected among lysine and arginine and combinations thereof. The polar uncharged amino acids of X3 are preferably selected among serine, threonine, asparagine and glutamine and combinations thereof.
The aromatic amino acids of X4 are preferably selected among phenylalanine and tryptophan and combinations thereof; the polar uncharged amino acids are preferably selected among serine, asparagine and glutamine and combinations thereof; and the hydrophobic aliphatic amino acids are preferably selected among leucine, alanine and glycine and combinations thereon.
The compound according to the present invention may comprise additional groups X2, X5 and X6 in which case it has general formula Ia
X1xe2x80x94X2xe2x80x94[(X3)a/(X4)b/(X5)c]xe2x80x94X6xe2x80x83xe2x80x83Ia
wherein: X1, X3 and X4 have the same meaning as above,
X2 is selected among 0-3 glycine residues and 0-2 xcex2-amino alanine molecules;
X5 is a compound of general formula II 
xe2x80x83wherein Z stands for a spacer group selected among alkane and alkene containing 1-5 carbon atoms, J stands for a functional group selected among amines, thiols, alcohols, carboxylic acids and esters, aldehydes and alkyl halides; U is a labeling group c standing for an integer of 0-10; and
X6 being 0; or being selected among X1 (as hereinbefore defined);
within the subunit [(X3)a/(X4)b/(X5)c] the groups X3, X4 and X6 may be located at various suitable places.
X2 serves as linker A, between X2 and the sub-unit [(X3)a/(X4)b] or between X2 and the sub-unit [(X3)a/(X4)b/(X5)c];
X5 serves as linker B between the sub-unit [(X3)a/(X4)b] and X6 or between the sub-unit [(X3)a/(X4)b/(X5)c] and X6; and
X6 serves as main anchoring domain B.
U as a labeling group for specific binding is advantageously selected among biotin and a group containing a substituent selected among a fluorescein, a radioisotope and a paramagnetic contrast agent; the fluorescein may be, for example, fluorescein isothiocyanate; the radioisotope may be selected among technetium, lead, mercury, thallium and indium; and the paramagnetic contrast agent may be any paramagnetic metal ion chelate, e.g. gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA).
X5 is advantageously a lysine residue substituted at the xcex1-amino group by a labeling group as above defined.
In case that X6 stands for a cysteine residue bound through a thioether bond to a prenyl group the cysteine carboxyl group can be either free or methylated.
Any of the above amino acids may be the L-, the D- or the DL isomer or the racemate.
The amino acid residues may also be residues of suitable synthetic amino acids.
A sequence of the compounds of general formulae I and Ia is:
Myristate-GGGKKKKKRFSFKKSFKLSGFSFKKNKKK (SEQUENCE ID NO. 1)-U, in which G=glycine, K=lysine, R=arginine, F=phenylalanine, S=serine, L=leucine, N=asparagine and U as hereinbefore defined.
A preferred compound of said sequence is:
Myristate-GGGKKKKKRFSFKKSFKLSGFSFKKNKKK (SEQUENCE ID NO. 1)-(biotin). (This compound is herein called xe2x80x9cNST301xe2x80x9d.)
Another sequence of the compounds of general formulae I and Ia is
Myristate-KKKKKRFSFKKSFKLSGFSFKKNKKK (SEQUENCE ID NO. 2)-U, wherein K, R, F, S, L, G, N and U have the same meaning as above.
A preferred compound of said sequence is:
Myristate-KKKKKRFSFKKSFKLSGFSFKKNKKK (SEQUENCE ID NO. 2)-(biotin). (This compound is herein called xe2x80x9cNST302xe2x80x9d.)
The present invention also consists in pharmaceutical compositions comprising as active ingredient a NST300 compound as defined above with reference to genera formulae I and Ia. (Whenever the NST300 compound is mentioned herein it refers to the appropriate compounds as defined in formulae I and Ia).
In a preferred embodiment the pharmaceutical composition comprises in addition to the NST1300 compound a pharmaceutically acceptable carrier.
The carriers may be selected among any suitable components, e.g. solvents; emulgators; excipients; talc; flavors; colors; etc. The pharmaceutical composition may comprise, if desired, also other pharmaceutically active compounds. The pharmaceutical compositions may be, e.g. tablets, capsules, solutions, emulsions, etc.
The pharmaceutical composition according to the present invention may comprise an additional pharmaceutically active compound.
The amount of the NST300 compound incorporated in the pharmaceutical composition may vary widely. The factors which have to be considered when determining the precise amount are known to those skilled in the art. Such factors include, inter aria, the pharmaceutical carrier being part of the composition, the route of administration being employed and the frequency with which the composition is to be administered.
The pharmaceutical composition may be administered by any of the known methods, inter alia, per os, intravenous, intrapertional, intramuscular or subcutaneous or topical administration.
The present invention further consists in the use of a NST300 compound or of a pharmaceutical composition comprising same in the preparation of a medicament, in particular for the treatment or prevention or prothrombotic states; advantageously for the treatment of disorders which are associated with excessive pro-coagulant activity, initiated or propagated by CMLA loss, such as arterial or venous thrombosis; sickle cell disease; thalassemia; antiphospholipid antibody syndrome; lupus erythematosus; shed membrane particles, (e.g. during cardiopulmonary bypass); apoptosis, etc.
The present invention also consists in a method for the treatment or prevention of prothrombotic states; advantageously for the treatment of disorders which are associated with excessive pro-coagulant activity, initiated or propagated by CMLA loss, such as arterial or venous thrombosis; sickle cell disease; thalassemia; antiphospholipid antibody syndrome; lupus erythematosus; shed membrane particles, (e.g. during cardiopulmonary bypass); apoptosis, etc. by a NST300 compound or by a pharmaceutical composition comprising same.
The present invention also consists in the use of a NST300 compound or of a pharmaceutical composition comprising same for the diagnosis of CMLA loss. Said use may be performed either in vitro or in vivo in accordance with the specific requirements. Said uses are especially:
a. use as a diagnostic agent for the detection and imaging of cell death, particularly of apoptosis, either in vitro or in vivo. The in vitro imaging is preferably performed with fluorescin; the in vivo imaging is preferably performed by a scan with an isotope or by MRI;
b. use as a diagnostic agent for thrombosis or for prothrombotic states; and
c. use as a diagnostic agent for pathophysiological states associated with apoptosis; e.g monitoring of response to anticancer treatments, diagnosis of disorders of inappropriate excessive apoptosis, monitoring of response to cytoprotective treatments, monitoring of graft survival following organ transplantation.
The present invention also consists in a diagnostic kit comprising a NST300 compound or a pharmaceutical comprising same for the performance of the diagnostic steps.
The present invention also consists in the use of a NST300 compound or of a pharmaceutical composition comprising same as a targeting agent, to target drugs to tissues inflicted by CMLA loss, preferably tissues the cells of which are inflicted by excessive apoptosis, or tissues in which thrombosis in association with CMLA loss takes place.
The present invention also consists in a method for targeting drugs to tissues in the body which are inflicted by CMLA loss, which method comprises the conjugation of a NST300 compound or a pharmaceutical composition comprising same with a drug to be targeted through an esteric bond. The NST300 compound directs the conjugate to regions of CMLA loss. Subsequently, naturally-occurring cleavage or the esteric bond by local tissue esterases allows the liberation of the targeted drug to act in said region. The tissues are in particular those tissues the cells of which are inflicted by excessive apoptosis or tissues in which thrombosis in association with CMLA loss takes place.
The present invention also consists in the use of NST300 compounds or of pharmaceutical compositions comprising same for basic research, in fields of research in which CMLA loss takes place, both in vitro and in vivo, inter alia, of cell cultures, preferably in basic research of apoptosis.
Moreover, the present invention further consists in a process for the preparation of a NST300 compound of general formula I by the following steps:
a. for the preparation of the sub unit [(X3)a/(X4)b] an xcex1-amine protected, c-terminal amino acid of said sequence is loaded on a solid support, the xcex1-amine protecting group is removed, and the peptide sequence is sequentially prepared;
b. for coupling to X1 the xcex1-amino protecting group is removed from the N-terminal amino acid, and X1 is then introduced into the peptide-resin under the same conditions as in step a; and
c. finally the peptide is cleaved from the solid support, purified and characterized.
NST300 compounds of general formula Ia comprising sub-unit [(X3)a/(X4)b/(X5)c)] are prepared according to step (a) above.
For the preparation of X5 and its coupling to a labeling group or to X6 an orthogonally protected amino acid is loaded on a solid support; the protecting group on the xcfx89-functional group is selectively removed; X6 or the labeling group of X5 is introduced into the amino acid-resin in the Presence of an appropriate coupling reagent or by using a pre-activation method.
The coupling agent may be HBTU/HOBT and the pre-activation method may be the formation of an ester, azide or an anhydride.
Step (a) may also be used for the integration of X5 (either coupled to a labeling group or coupled to X6) into the peptide sequence.
The characterization is preferably performed by high performance liquid chromatographyxe2x80x94mass spectra (HPLC-MS)
Step (a) and the pre-activating may be performed on the basis of knowledge of solid phase peptide synthesis (Atherton E, Sheppard R C, Solid phase peptide synthesis; a practical approach, IRL Press, 1989; Bodanszky M, Peptide Chemistry, Springer Verlag, 1988.)